The influence of DNA double-strand break structure on end-joining in human cells.

DNA end-joining is the major repair pathway for double-strand breaks (DSBs) in higher eukaryotes. To understand how DSB structure affects the end-joining process in human cells, we have examined the in vivo repair of linearized plasmids containing complementary as well as several different configurations of non-complementary DNA ends. Our results demonstrate that, while complementary and blunt termini display comparable levels of error-free rejoining, end-joining fidelity is decreased to varying extents among mismatched non-complementary ends. End structure also influences the kinetics of repair, accurately recircularized substrates for blunt and complementary termini being detected significantly earlier than for mismatched non-complementary ends. These results suggest that the end-joining process is composed of an early component, capable of efficiently repairing substrates requiring a single ligation event, and a late component, involved in the rejoining of complex substrates requiring multiple processing steps. Finally, these two types of repair events may have different genetic requirements as suggested by the finding that exposure of cells to wortmannin, a potent inhibitor of phosphatidylinositol 3-related kinases (PI 3-related kinases), blocks the repair of complex substrates while having little or no effect on those requiring a simple ligation event.

Arrest of S-phase progression is impaired in Fanconi anemia cells.

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability, hypersensitivity to DNA cross-linking agents, and a prolonged G2 phase of the cell cycle. We observed a marked dose-dependent accumulation of FA cells in the G2 compartment after treatment with 4,5',8-trimethylpsoralen (Me(3)Pso) in combination with 365 nm irradiation. Using bivariate DNA distribution methodology, we determined the proportion of replicating and arresting S-phase cells and observed that, whereas normal cells arrested DNA replication in the presence of Me(3)Pso cross-links and monoadducts, FA lymphoblasts failed to arrest DNA synthesis. Taken together, the above data suggest that, in response to damage induced by DNA cross-linking agents, the S-phase checkpoint is inefficient in FA cells. This would lead to accumulation of secondary lesions, such as single- and double-strand breaks and gaps. The prolonged time in G2 phase seen in FA cells therefore exists in order to allow the cells to remove lesions which accumulated during the preceding abnormal S phase.

Molecular spectra of HPRT deletion mutations in circulating T-lymphocytes in Fanconi anemia patients.

The principal cellular feature of Fanconi anemia (FA), an inherited cancer prone disorder, is a high level of chromosomal breakage, amplified after treatment with crosslinking agents. Three of the eight genes involved in FA have been cloned: FANCA, FANCC and FANCG. However, their biological functions remain unknown. We previously observed an excessive production of deletions at the HPRT locus in FA lymphoblasts belonging to the relatively rare complementation group D(1) and an increased frequency of glycophorin A (GPA) variants in erythrocytes derived from FA patients (2). In thi study, we examined the molecular nature of 31 HPRT mutations formed in vivo in circulating T-lymphocytes isolated from 9 FA male patients. The results show that in all FA patients investigated the deletions are by far the most prevalent mutational event in contrast to age matched healthy donors, in which point mutations predominate. The complementation group in the FA patients examined in the present study has not yet been defined. However, knowing that mutations in the FANCA and FANCC gene are found to be involved in at least 70% of the FA patients, it can be expected that the excessive production of deletions is a general feature of the FA phenotype. In addition, the spectrum of HPRT deletions observed in FA patients differs from that of healthy children: there is a high frequency of 3'-terminal deletions and a strikingly low proportion of V(D)J mediated events. Based on previous findings, a decreased fidelity of coding V(D)J joint formation (3) and an inaccurate repair of specific DNA double strand breaks via Non-Homologous End Joining (4), we propose that FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion pronenness.

A human severe combined immunodeficiency (SCID) condition with increased sensitivity to ionizing radiations and impaired V(D)J rearrangements defines a new DNA recombination/repair deficiency.

The products of recombination activating gene (RAG)1 and RAG2 initiate the lymphoid-specific phase of the V(D)J recombination by creating a DNA double-strand break (dsb), leaving hairpin-sealed coding ends. The next step uses the general DNA repair machinery of the cells to resolve this dsb. Several genes involved in both V(D)J recombination and DNA repair have been identified through the analysis of in vitro mutants (Chinese hamster ovary cells) and in vivo situations of murine and equine severe combined immunodeficiency (scid). These studies lead to the description of the Ku-DNA-dependent protein kinase complex and the XRCC4 factor. A human SCID condition is characterized by an absence of B and T lymphocytes. One subset of these patients also demonstrates an increased sensitivity to the ionizing radiation of their fibroblasts and bone marrow precursor cells. This phenotype is accompanied by a profound defect in V(D)J recombination with a lack of coding joint formation, whereas signal joints are normal. Functional and genetic analyses distinguish these patients from the other recombination/repair mutants, and thus define a new group of mutants whose affected gene(s) is involved in sensitivity to ionizing radiation and V(D)J recombination.

Abnormal rearrangements associated with V(D)J recombination in Fanconi anemia.

The hallmark of Fanconi anemia (FA), a rare inherited cancer prone disorder, is a high level of chromosome breakage, spontaneous and induced by cross-linking agents. The increased genomic instability of FA is reflected at the gene level by an overproduction of intragenic deletions. Two of the eight FA genes have been cloned, however, their function remains unknown. We recently demonstrated that the lack of functional FA genes lead to a marked decrease in the fidelity of non-homologous end-joining, a pathway that mammalian cells predominantly use to repair DNA double-strand breaks (DSB). Knowing that specific DSB are generated during V(D)J recombination, here we have examined the molecular features of V(D)J rearrangements in normal and FA lymphoblasts belonging to complementation groups C and D. Using appropriate extrachromosomal recombination substrates, V(D)J coding and signal joint formation have been analysed quantitatively and qualitatively. Our results show that the frequency of coding and signal joint formation was not significantly different in normal and FA cells. However, when the fidelity of the V(D)J reaction was examined, we found that in normal human lymphoblasts V(D)J recombination proceeds with high precision, whereas, in FA cells a several fold increase in the frequency of aberrant rearrangements is associated with V(D)J coding joint formation. The abnormal recombinants that we recovered in FA are consistent with excessive degradation of DNA ends generated during the V(D)J reaction. On the basis of these findings, we propose a working model in which FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion proneness.

Fanconi anemia C gene product plays a role in the fidelity of blunt DNA end-joining.

Mutations in genes controlling the correct functioning of the replicative, repair and recombination machineries may lead to genomic instability. A high level of spontaneous chromosomal aberrations amplified by treatment with DNA cross-linking agents is the hallmark of Fanconi anemia (FA), an inherited chromosomal instability syndrome associated with cancer proneness. Two of the eight FA genes have been cloned (FAA and FAC), but their function has not yet been defined. The lack of homology with known genes suggests the involvement of FA genes in a novel pathway specific to vertebrates. Using a DNA end-joining assay in cultured cells, we studied the processing of both blunt and cohesive-ended double strand breaks (DSB) in normal and FA cells. The results show that: (i) the overall ligation efficiency is normal in FA lymphoblasts; (ii) in FA-C, error-free processing of blunt-ended DSB is markedly decreased, resulting in a higher deletion frequency and larger deletion size; (iii) the fidelity of processing of blunt-DSB is completely restored in FACC cells (complemented with wild-type FAC gene) and the deletion size shifted to values similar to that observed in normal cells; (iv) the fidelity of cohesive end-joining is not affected in FA cells; (v) activities and/or expression of known factors involved in DSB processing, such as the components of the DNA-PK complex and XRCC4, are normal in FA cells. Our results provide strong evidence that the lack of a functional FAC gene results in loss of fidelity of end-joining, which likely accounts for the FA-C phenotype of chromosome instability. We conclude that FAC, and perhaps all FA gene products, are likely to play a role in the fidelity of end-joining of specific DSB.

The fidelity of double strand breaks processing is impaired in complementation groups B and D of Fanconi anemia, a genetic instability syndrome.

In mammalian cells, nonhomologous end-joining is the predominant mechanism to eliminate DNA double strand breaks. Such events are at the origin of deletion mutagenesis and chromosomal rearrangements. The hallmark of Fanconi anemia, an inherited cancer prone disorder, is increased chromosomal breakage associated to over-production of deletions. Knowing that double strand breaks are at the origin of deletion mutagenesis, the question arises whether their processing is affected in FA. We set up a "host cell end-joining assay" to analyze the fate of double strand breaks into extrachromosomal substrates transiently replicated in normal and FA-D lymphoblasts. Although no difference in plasmid survival was found, blunt-ended breaks were sealed with significantly lower fidelity in FA cells, resulting in a higher deletion frequency and a larger deletion size. The results suggest that FA-D and FA-B gene products are likely to play a role in end-joining fidelity of specific DNA double strand breaks.

Lack of detectable defect in DNA double-strand break repair and DNA-dependent protein kinase activity in radiosensitive human severe combined immunodeficiency fibroblasts.

The initial step of the V(D)J recombination occurs through the generation of a DNA double-strand break (dsb). Defects in the DNA-dependent protein kinase complex (DNA-PK) result in an inability to perform either V(D)J recombination or any dsb repair effectively. The human autosomal T-B-severe combined immunodeficiency (SCID) condition is characterized by an absence of both B and T lymphocytes and is accompanied in some patients by an increase in gamma-ray sensitivity (T-B-RS SCID) comparable to that found in mouse SCID cells. We show here that cells from six patients with T-B-RS SCID had normal DNA-dsb repair kinetics. Furthermore, DNA-PK activity was present in extracts from these human T-B-RS SCID fibroblasts. We therefore conclude that some human T-B-RS SCID disorders are not caused by a defect in an essential DNA-PK component.

The mutagenic processing of psoralen photolesions leaves a highly specific signature at an endogenous human locus.

To assess the role of a given genotoxic agent in the etiology of human cancers, it is useful to establish the mutational specificity of this agent. The aim of this study was to investigate whether the processing of psoralen photolesions, interstrand cross-links (CL) and monoadducts (MA), leaves a specific molecular signature in the mutational events produced at an endogenous locus, HPRT. Human lymphoblasts were treated by 4,5',8-trimethylpsoralen (Me3Pso) in association with a double irradiation protocol (365 plus 365 nm) which allows us to increase the proportion of CL for a given constant number of total photoadducts. The molecular spectrum of mutations at the HPRT locus induced in these conditions was compared to the previously reported spectra of mutations induced by the same psoralen in combination with a single irradiation of either 365 nm (induction of MA and a low proportion of CL) or 405 nm (producing almost exclusively MA). In all treatment conditions, base substitutions constitute the major type of Me3Pso photoinduced mutations. The majority of base substitutions involve a T residue preferably within a 5'-TpA sequence which corresponds to the favoured sites of psoralen photoadducts. In other words, the Me3Pso photolesions induce at the endogenous HPRT locus a high specific signature. Moreover, base substitutions have been essentially found in the non-transcribed strand of the HPRT gene suggesting that the psoralen photolesions are preferentially removed from the transcribed strand. In spite of the considerable difference between the proportion of lesions of both types (CL or MA) induced in different treatment conditions, the kind of mutations and their sequence distribution are similar suggesting that the mutagenic processing of psoralen CL and MA is similar at least for the steps resulting in base substitutions.

Genotoxic potential of psoralen cross-links versus monoadducts in normal human lymphoblasts.

Using the 4,5',8-trimethylpsoralen in combination with the reirradiation protocol, we show that, in normal human lymphoblasts, the cytotoxic potential of photoinduced cross-links (CL) is higher than that of monoadducts (MA). In contrast to cytotoxicity, the significant increase in the proportion of CL, at a constant level of total adducts, had no effect on the induction of mutations at the HPRT locus. Comparison with the data obtained in yeast and rodent cells using the same double irradiation protocol shows that the mutagenic potential of CL versus MA varies between species. This suggests that the equilibrium between the excision, the recombinational and the mutagenic components of the repair pathways which probably determine the mutagenic efficiency of CL versus MA is likely to be species-dependent.

The molecular mechanism underlying formation of deletions in Fanconi anemia cells may involve a site-specific recombination.

Spontaneous and induced chromosomal breakage is an important cellular feature of Fanconi anemia (FA), an inherited DNA repair disorder characterized by progressive bone marrow failure, developmental abnormalities, and predisposition to leukemia. We have previously reported that in comparison to normal cells, there is a substantial increase in frequency of intragenic deletions at an endogenous locus (HPRT) in FA lymphoblasts. Taken together with the increased chromosomal instability, these observations indicated that the wild-type FA gene(s) plays an important role in the maintenance of the genomic integrity. To obtain information on the mechanism(s) underlying the genomic rearrangements in FA, the breakpoint sites of deletions in 11 FA-derived HPRT- mutants were analyzed. The results indicate that a significant proportion of deletions involving a loss of a given exon are identical and that two deletions of different size have the same 3' breakpoint. Interestingly, it appears that in most of the mutants there is a common deletion signal sequence, which suggests that the mutations in the FA gene(s) may lead to an aberrant site-specific cleavage activity that might be responsible for the deletion proneness and the chromosomal instability characteristic of the FA pathology. From the similarity or even identity of the signal sequence at some of the breakpoints with the consensus heptamer which directs cleavage and joining in the assembly of immunoglobulin and T-cell receptor genes, we speculate that steps in common with the V(D)J recombinational process may be illegitimately involved in FA cells.

Frequencies of HPRT- lymphocytes and glycophorin A variants erythrocytes in Fanconi anemia patients, their parents and control donors.

The mutant frequency of 6-thioguanine resistance (HPRT locus) in circulating T lymphocytes from 23 Fanconi anemia (FA) patients has been determined. The glycophorin A (GPA) in vivo cell mutants assay, which detects allele loss variant phenotypes arising from mutations in erythroid progenitor cells of GPA heterozygous MN individuals, has been applied in parallel to FA patients. No significant difference in frequency of HPRT- mutants was observed in FA compared to age matched healthy donors. In contrast, the mean frequency of GPA variant cells was elevated 31-fold for hemizygous NO variants and 8-fold for homozygous NN variants in FA patients over normal controls. In heterozygous FA parents, HPRT- mutant frequencies and GPA variant frequencies were within the normal range. Molecular analysis of HPRT- mutants has previously shown that FA cells have a high tendency to form deletions. Knowing that the cellular events allowing the detection of mutations at the HPRT and the GPA locus differ, our results emphasize the possible correlation between events of spontaneous loss of heterozygosity and genetic predisposition to cancer as observed in FA.

Molecular spectrum of mutations induced at the HPRT locus by a cross-linking agent in human cell lines with different repair capacities.

Molecular characterization of mutations photoinduced by a cross-linking agent, 4,5',8-trimethylpsoralen (Me3Pso), in normal human lymphoblasts was conducted in parallel with lymphoblasts derived from Fanconi anemia patients. Such cells have been previously described to be impaired in repair of psoralen photolesions. The endogenous HPRT locus was used as a target gene. The treatment of cells with Me3Pso in combination with 365 nm irradiation leads to the formation of interstrand cross-links, and specific monoadducts. Our analysis revealed that the mutagenic processing of Me3Pso photoadducts in normal human cells results essentially in base substitutions (84%). These are localized to sequences shown previously to be favored for the formation of Me3Pso monoadducts. The mutagenic processing of the same lesions in Fanconi anemia cells results in fewer base substitutions (22%), with deletions (66%) being the predominant class of mutation. In contrast to prokaryotic systems, frameshifts are poorly represented among Me3Pso induced mutations in human cells. In spite of important differences between the kinds of mutations observed in the two cell lines, our analysis reveals similarities in the type of base substitutions and their sequence distribution. In both normal and Fanconi anemia cell lines mutations, mostly targeted on thymine residues, are preferentially located on the non-transcribed strand.

Mutagenic processing of psoralen monoadducts differ in normal and Fanconi anemia cells.

The molecular spectra of mutations photoinduced (405 nm) by 4,5',8-trimethylpsoralen monoadducts (MA), at an endogenous locus, hypoxanthine-guanine phosphoribosyl-transferase (HPRT) in normal and in a Fanconi anemia (FA) lymphoblast cell line, complementation group D, are presented. We show that, in normal cells, MA induce only base substitutions. In contrast, in FA cells which are partially deficient in the incision of MA, deletions are preferentially induced over point mutations (62% of the total). Although the proportion of base substitutions is lower in FA cells, their type and sequence distribution are similar in FA and normal cell lines. The majority of base substitutions are located at sites of psoralen MA which suggest that 4,5',8-trimethylpsoralen photoinduced mutations are targeted and preferentially formed in the non-transcribed strand. Moreover, point mutations induced by MA in normal and FA cells are not homogeneously distributed, they preferentially occur in exon 8 of the HPRT gene. This heterogeneous distribution of mutations is ascribed to processing of MA. Great similarities were found between normal and FA cells with respect to the nature and location of point mutation at the HPRT gene; the high proneness to deletions remains one of the major instability features of FA.

Increased radiosensitivity of granulocyte macrophage colony-forming units and skin fibroblasts in human autosomal recessive severe combined immunodeficiency.

We studied the radiosensitivity of granulocyte macrophage colony-forming units (GM-CFU) in patients with a severe combined immunodeficiency (SCID). Three patients lacking both mature T and B cells showed a twofold higher GM-CFU radiosensitivity calculated as the DO value (dose required to reduce survival to 37%), and an identical observation was made with fibroblasts from one of these patients. A patient with an SCID with hypereosinophilia, i.e., Omenn's syndrome characterized by extremely restricted T cell heterogeneity and a lack of B cells, also showed abnormal GM-CFU radiosensitivity. In contrast, GM-CFU from a patient lacking only T cells (X-linked form of SCID) showed normal GM-CFU radiosensitivity. These data further support the similarity between human T(-) B(-) SCID and the murine acid mutation characterized by a defect in T cell receptor and immunoglobulin gene rearrangement, and by an abnormal double-strand DNA break repair function. In addition, they strongly suggest that the Omenn's immunodeficiency syndrome may be a leaky T(-)B(-) SCID phenotype as previously indicated by the coexistence of the two phenotypes in siblings.

Decreased deletion mutation in radioadapted human lymphoblasts.

Survival and HPRT- mutant frequency were measured in human lymphoblastoid cells preexposed or not to a low dose of 0.02 Gy gamma rays and then treated with a high dose of 4.0 Gy. When compared to the high dose alone, the low-dose preexposure induced a 70% reduction of the mutant frequency, whereas cell survival was not affected. To understand the mechanisms underlying this phenomenon, the molecular nature of an HPRT- mutant collection was established using Southern hybridization analysis. Among mutants induced by irradiation with the high dose alone, 78% (21/27) had detectable alterations of the HPRT gene. In contrast, only 42% (15/26) of mutants which were "adapted" by the low-dose exposure were of this type. Moreover, the fraction of mutants with only partial deletions of the HPRT gene was significantly reduced (52 and 19% for the 4.0 Gy and 0.02 Gy plus 4.0 Gy induced mutant sets, respectively). In other words, the mutational specificity differed, depending on whether or not the cells were adapted. We suggest that low-dose preexposure leads to a reduced susceptibility to the mutagenic effect of a high dose of gamma rays by inducing an error-free repair system. Our data indicate that this putative system acts preferentially on the class of premutagenic lesions which produce deletions.

HPRT gene expression differs in mutants derived from normal and Fanconi anemia cells: analysis of spontaneous and psoralen-photoinduced mutants.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by chromosomal instability and abnormalities in the processing of DNA lesions induced by cross-linking agents. We previously reported that after photoaddition of psoralen derivatives the frequency of HPRT- mutants was significantly lower in FA than in normal human lymphoblasts. The hypomutability in FA cells was shown to be associated with an increased deletion frequency at the HPRT gene level. Further characterization of 70 unrearranged mutants (without detectable changes in restriction enzyme fragment length) according to the HPRT gene expression is reported here. Northern blot hybridization analysis demonstrates considerable differences in mRNA phenotyping between normal and FA cells. In normal cells, the minority of spontaneous (31%) and psoralen-induced mutants (0% and 14% according to treatment) arise from mutations that alter the HPRT gene transcription. In contrast to normal cells, in the majority of mutants isolated from FA cells, HPRT gene expression is found to be affected. Indeed a large proportion of either spontaneous (67%) or psoralen-induced (56% and 46%) mutants did not produce detectable amounts of mRNA. These results suggest that the mutagenic processing of spontaneous and psoralen-photoinduced lesions differs in normal and FA cells.

Mutagenic effects photoinduced in normal human lymphoblasts by a monofunctional pyridopsoralen in comparison to 8-methoxypsoralen.

The photobiological effects induced by the monofuctional 7-methylpyrido[3,4-c]psoralen (MePyPs) in comparison to the bifunctional furocoumarin 8-methoxypsoralen (8-MOP) have been studied in a human lymphoblast cell line TK6. We report that, in human lymphoblasts, the cytotoxic effect of MePyPs plus UVA (365 nm) is much higher than that of 8-MOP plus 365-nm irradiation. The dose-modifying factor at the 37% survival level between the 2 compounds equals 120. Mutation induction by photoactivated MePyPs and 8-MOP has been studied in 2 genetic loci, hypoxanthine phosphoribosyl transferase (HPRT) and Na+/K+ ATPase. For equal UVA doses, the mutagenic effectiveness of MePyPs was higher than that of 8-MOP. However at equal survival levels, the mononfuctional psoralen MePyPs was less efficient than the bifunctional 8-MOP. In other words, compared to 8-MOP, the monofunctional agent MePyPs is more cytotoxic than mutagenic. This higher phototoxic and mutagenic efficiency of MePyPs in comparison to 8-MOP is likely to be related to the chemical nature of MePyPs-induced lesions which may be responsible for a reduced recognition and/or accessibility of the repair enzymes to damaged DNA.

Hypomutability in Fanconi anemia cells is associated with increased deletion frequency at the HPRT locus.

Fanconi anemia (FA) is an inherited human disorder associated with a predisposition to cancer and characterized by anomalies in the processing of DNA cross-links and certain monoadducts. We reported previously that the frequency of psoralen-photoinduced mutations at the HPRT locus is lower in FA cells than in normal cells. This hypomutability is shown here to be associated with an increased frequency of deletions in the HPRT gene when either a mixture of cross-links and monoadducts or monoadducts alone are induced. Molecular analysis of mutants in the HPRT gene was carried out. In normal cells the majority of spontaneous and induced mutants are point mutations whereas in FA deletion mutations predominate. In that case a majority of mutants were found to lack individual exons or small clusters of exons whereas in normal cells large (complete or major gene loss) and small deletions are almost equally represented. Thus we propose that the FA defect lies in a mutagenic pathway that, in normal cells, involves bypassing lesions and subsequent gap filling by a recombinational process during replication.

Mutagenic response of Fanconi's anemia cells from a defined complementation group after treatment with photoactivated bifunctional psoralens.

The induction of mutants at the hypoxanthine-guanine phosphoribosyltransferase and Na+/K+ ATPase loci by photoaddition of two bifunctional psoralens was compared in normal and in Fanconi's anemia lymphoblasts from the genetic complementation group A. For the two loci, the frequency of mutants was significantly lower in Fanconi's anemia than in normal cells. This is true whether the data are expressed as a function of dose or as a function of survival level. It is suggested that the chromosomal instability characteristic of Fanconi's anemia is responsible for the cancer proneness rather than the mutability at the gene level.